THE MOLECULAR MECHANISM AND POTENTIAL DEPENDENCE OF THE NA+ GLUCOSE COTRANSPORTER/

Activity of the Na+/glucose cotransporter endogenously expressed in LL C-PK1 cells was measured using whole cell recording techniques under t hree different sodium concentration conditions: 1) externally saturati ng, zero trans; 2) 40 mM external, zero trans; and 3) externally satur ating, 50 mM trans. Activity of the transporter with increasing concen trations of sugar was measured for each set of conditions, from which the maximal current for saturating sugar, I-m was determined. The I-m measured shows substantial potential dependence for each set of condit ions. The absolute I-m and the relative potential dependence of I-m co mpared among the various solute conditions were used to identify which loci in the transport cycle are responsible for potential-dependent c hanges in function. The experimental data were compared with the predi cted I-m values calculated from an eight-state, sequential, reversible model of a transport reaction kinetic scheme. Predictions derived fro m assignment of rate limitation and/or potential dependence to each of the 10 transitions in the transport pathway were derived and compared with the measured data. Most putative models were dismissed because o f lack of agreement with the measured data, indicating that several st eps along the transport pathway are not rate limiting and/or not poten tial dependent. Only two models were found that can completely account for the measured data. In one case, translocation of the free carrier must be rate limiting, and both extracellular sodium-binding events a s well as translocation of both free and fully loaded carrier forms mu st be potential-dependent transitions. In the second case, translocati on of the free carrier and dissociation of the first sodium to be rele ased intracellularly must be equivalently rate limiting. In this case only the two translocation events are required to be potential depende nt. The two external sodium-binding events might still be potential de pendent, but this is not required to fit the data. Previous reports su ggest that the first model is correct; however, no direct experimental data compel us to dismiss the second option as a feasible model.